Most myocardial infarctions are caused by coronary thrombosis (DeWood et al., N. Eng. J. Med. 303:897 (1983). The coronary thrombosis that causes the myocardial infarction can be lysed by thrombolytic agents. These thrombolytic agents are plasminogen activators that activate the conversion of plasminogen to the fibrinolytic enzyme plasmin. Plasmin will then lyse the fibrin present in the thrombus. This treatment with plasminogen activators is not without side effects. Plasmin acts non-selectively and therefore, not only lyses the fibrin in the thrombus, but also attacks fibrinogen and clotting factors, often resulting in severe bleeding diathesis.
Streptokinase, urokinase and tissue-type plasminogen activator (TPA) are three known plasminogen activators for lysing thrombi. These activators are indicated for the treatment for acute cardiovascular disease such as infarct, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, arid other venous thrombosis. Both streptokinase and urokinase, however, have severe limitations. Due to a low affinity for fibrin, both activators will activate circulating and fibrin-bound plasminogen indiscriminately. The plasmin formed in circulating blood is neutralized before it can be used in thrombolysis. Residual plasmin will degrade several clotting factor proteins, for example, fibrinogen, factor V, and factor VIII, causing hemorrhagic potential. Further, streptokinase is strongly antigenic and patients with high antibody titers respond inefficiently to treatment and cannot remain on continuous treatment.
Human tissue-type plasminogen activator can bind to fibrin and therefore favors the activation of plasminogen in close proximity to the thrombus, potentially sparing fibrinogen elsewhere in the circulation. However, at doses required for prompt lysis of coronary thrombi, the use of tissue-type plasminogen activator can also result in hemorrhage.
In order to increase the specificity of the thrombolytic agents to the thrombus, it has been shown that covalent linkage of urokinase to a fibrin-specific antibody results in marked enhancement of fibrinolytic potency and specificity Bode et al., Science 229:765-767 (1985).
One function characteristic of every antibody molecule is specific binding to an antigenic determinant. Antibodies in vivo are bivalent and monospecific, containing two identical antigen binding sites. The specific binding of antigen by an antibody molecule is determined by the antibody's structure of the variable regions (F.sub.ab) of both heavy and light chains. Antibodies having dual specificities have been prepared by subjecting antibodies of different specificities to a selective cleavage of the disulfide bridges that link the two heavy chains together. Antibody half-molecules are then reassociated under neutral pH to produce the hybrid antibodies having dual specificities.
Nisonhoff et al., Nature (London) 194:355 (1962), describe the in vitro production of a bispecific antibody molecule from a polyclonal rabbit antibody, anti-ovalbumin, and an anti-bgg antibody. The monospecific antibodies were treated with pepsin to remove the F.sub.c portion of the antibody, leaving the two antigen-binding sites (F.sub.ab) covalently linked by a single disulfide bond. This bond was then split under reducing conditions and the two antibody molecules reassociated under oxidizing conditions to produce a bispecific antibody.
In Brennan et al., Science 299:31 (1985), a chemical procedure is described for preparing bispecific antibody fragments from monoclonal antibodies. In this procedure, a modification of the Nisonoff technique was used in cleaving the F.sub.ab fragments, followed by reconstituting the half-fragments to form the bispecific antibody molecule. The F.sub.ab fragments were reduced in the presence of sodium arsenite to stabilize vicinal dithiols and impede intramolecular disulfide formation. The other modification involved activating the thiols of one of the half-F.sub.ab fragments as a thionitrobenzoate derivative. By this process, a bispecific antibody was produced from anti-avidin F.sub.ab and anti-luciferase F.sub.ab was produced.
Liu et al., Proc. Natal. Acad. Sci. USA 82:8648 (1985), disclose a chemical procedure for forming a bispecific antibody in which anti-T3 antibody was covalently linked to a second monoclonal antibody, anti-IgId specific for the idiotype of the surface immunoglobulin of a human B lymphoma. The anti-T3 and anti-IgId antibodies were first reacted with N-suc-cinimidyl-3-(2-pyridyldithio) propionate (SPDP). Thiol groups were attached to the cleaved anti-T3 antibody using 2-iminothiolane. Then the two modified half-antibodies, anti-T3 and anti-IgId, were mixed to covalently link the two antibodies. The result showed that the T8 cytotoxic T lymphocytes lysed the human B-lymphoma cells, but no lyses was observed when T4 cytotoxic T lymphocyte cells were used.
Bispecific antibodies have also been produced from hybridomas. The preparation of bispecific monoclonal antibodies by fusion of antibody-producing hybridoma cells is described in Milstein and Cuello, Nature (London) 305:537 (1983). This reference describes the fusion of two hybridomas, or the fusion of one hybridoma with spleen cells from an immunized rat, to produce hybrid hybridomas. These hybrid hybridomas secrete predefined bispecific monoclonal antibodies as well as monospecific antibodies. Anti-somatostatin/anti-pluroxidase and anti-substance P/anti-peroxidase bispecific monoclonal antibodies were prepared in this manner. The bispecific monoclonal antibodies produced by hybrid hybridomas were complete molecules, containing the F.sub.c region as well as the antigen-combining sites.
PCT application, WO83/03679, describes the production of a bispecific antibody having dual specificities obtained by fusion two hybridomas. This application describes procedures for producing and selecting hybrid hybridomas. The bispecific antibodies therein are described as having many potential uses, ranging from immunodiagnostic procedures to targeted delivery of drugs.
It would be desirable to have a bispecific antibody having dual specificity such that one specificity would be directed against a thrombus and the other specificity would be directed against a thrombolytic agent. With this bispecific antibody, a thrombus would be detected. This thrombus then could be lysed by the action of a thrombolytic agent that becomes or is attached to the anti-thrombolytic antibody. The lysis of thrombi is complicated; it was not known whether a bispecific antibody would block or inhibit the thrombolytic activity by the thrombolytic agent.